Cholinergic modulation of the basal L-type calcium current in ferret right ventricular myocytes

The effects of the cholinergic muscarinic agonist carbachol (CCh) on the basal L-type calcium current, I(Ca,L), in ferret right ventricular (RV) myocytes were studied using whole cell patch clamp. CCh produced two major effects : (i) in all myocytes, extracellular application of CCh inhibited I(Ca,L) in a reversible concentration-dependent manner; and (ii) in many (but not all) myocytes, upon washout CCh produced a significant transient stimulation of I(Ca,L) ('rebound stimulation'). Inhibitory effects could be observed at 1 x 10(-10) M CCh. The mean steady-state inhibitory concentration-response relationship was shallow and could be described with a single Hill equation (maximum inhibition = 34.5 %, IC50 = 4 x 10(-8) M, Hill coefficient n = 0.60). Steady-state inhibition (1 or 10 microM CCh) had no significant effect on I(Ca,L) selectivity or macroscopic (i) activation characteristics, (ii) inactivation kinetics, (iii) steady-state inactivation or (iv) kinetics of recovery from inactivation. Maximal inhibition of nitric oxide synthase (NOS) activity (preincubation of myocytes in 1 mM L-NMMA (N(G)-monomethyl-L-arginine) + 1 mM L-NNA (N(G)-nitro-L-arginine) for 2-3 h plus inclusion of 1 mM L-NMMA + 1 mM L-NNA in the patch pipette solution) produced no significant attenuation of the CCh-mediated inhibition of I(Ca,L). Protocols involving (i) the nitric oxide (NO) scavenger PTIO (2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide; 200 microM), (ii) imposition of a 'cGMP clamp' (100 microM 8-Bromo-cGMP), and (iii) inhibition of soluble guanylyl cyclase (ODQ (1H-[1,2,4,]oxadiazolo(4,3,-a)quinoxalin-1-one), 50 microM) all failed to attenuate CCh-mediated inhibition of I(ca,L). While CCh consistently inhibited basal I(Ca,L) in all RV myocytes studied, not all myocytes displayed rebound stimulation upon CCh washout. However, there was no difference between CCh-mediated inhibition of I(Ca,L) between these two RV myocyte types, and in myocytes displaying rebound stimulation neither ODQ nor 8-Bromo-cGMP (8-Br-cGMP) altered the effect. We conclude that NO production, activation of soluble guanylyl cyclase, or changes in intracellular cGMP levels are not obligatorily involved in muscarinic-mediated modulation of basal I(Ca,L) in ferret RV myocytes.